Background
Significant evidence has shown that the miRNA pathway is an important component in the downstream signaling cascades of TGF-β1 pathway. Our previous study has indicated that miR-335-5p expression was significantly down-regulated and acted as a vital player in the metastasis of non-small cell lung cancer (NSCLC), however the underlying mechanism remained unclear.
Conclusion
This is the first time to report that miR-335-5p regulates ROCK1 and impairs its functions, thereby playing a key role in TGF-β1-induced EMT and cell migration and invasion in NSCLC.
Methods
The differential expression level of miR-335-5p and ROCK1 were determined by qRT-PCR and IHC analysis in human tissue samples with or without lymph node metastasis. Transwell assay was conducted to determine cell ability of migration and invasion. SiRNA interference, microRNA transfection and western blot analysis were utilized to clarify the underlying regulatory mechanism.
Results
We showed that down-regulated expression of miR-335-5p and up-regulated expression of ROCK1 in NSCLC tissues were associated with lymph node metastasis. Over-expresion of miR-335-5p significantly inhibited TGF-β1-mediated NSCLC migration and invasion. Furthermore, luciferase reporter assays proved that miR-335-5p can bind to 3'-UTR of ROCK1 directly. Moreover, we confirmed that siRNA-mediated silencing of ROCK1 significantly diminished TGF-β1-mediated EMT and migratory and invasive capabilities of A549 and SPC-A1 cells.